The invention relates to the regeneration of a damaged mammalian nerve and particularly to a method and apparatus for in vivo mammalian nerve regeneration using an electric potential gradient or electric current established from the proximal nerve end (that is, the nerve end closest to the cell body) to the distal nerve end (that is, the nerve end furthest from the cell body) with the nerve ends either spaced apart from each other or sutured together.
Considerable research relating to nerve growth and nerve regeneration has produced numerous publications relating to these topics. The distinction between "nerve growth" and "nerve regeneration" is significant. The growth of a nerve does not assure that the nerve will function even partially as it did prior to damage. That is, "nerve growth" does not assure the functioning of the nerve as a channel for communication of information. In contrast, "nerve regeneration" as used herein is the regeneration of a nerve to serve at least partially as part of the communication system of the nervous system. It is well known in the art that there are substantial differences between a mammalian nervous system and other nervous systems. Thus, the evaluation of method and apparatuses for mammalian nerve regeneration must be carried out on a mammal to have any value and credibility.
When axons of the mammalian peripheral nervous system (PNS) are severely damaged (i.e., in compression or transection injuries), several phenomena may take place. First, if left unmodified, the distal stump will nearly always degenerate (Wallerian degeneration). This degeneration is associated with concomitant chromatolytic changes proximally in the perikaryon. If damage to the nerve is sufficiently severe, the axons in the proximal portion will degenerate, followed by the degeneration and (usually) death of the perikaryon. If the damage to the nerve is less severe and left unmodified, a number of biochemical changes begin to occur in the remaining proximal portion. These proximal changes involve a complex series of responses of the cell body to the injury which seem to prepare the intact portion for regeneration. Such changes include alterations in axonal transport characteristics, protein processing and nucleic acid synthesis. Morphologically regenerating growth cones from the damaged proximal stump often appear, and the axons will begin to regenerate from the proximal stump towards the original target region.
Such damage is likely to produce collateral sprouting from neighboring axons that are not as severly injured and which did not undergo Wallerian degeneration. The newly growing neurites probably use the degenerated distal segment as a guide to the denervated target area. Presumably, the reactive Schwann cells provide the communicative means for regeneration to the target by providing diffusable growth-promoting factors, and by providing a suitable growth surface established by the plasma membrane and/or basal lamina, or extracellular matrix. All of these morphological and biochemical changes are primarily dependent upon several factors, including the severity and location of the injury depending upon the proximity to the perikaryon, the size of the axons injured, and the species involved. For example, higher vertebrates have less capacity to regenerate the peripheral nervous system (PNS) axons effectively.
In the central nervous system (CNS), the unaided attempts at regeneration often are quickly aborted by the body, resulting in a completely degenerated proximal stump and perikaryon. Recent publications have improved the understanding of the cellular events in the CNS following injury. Transplantation of embryonic tissues into the brain or spinal cord has proven to be a useful tool for determining the essential factors for regeneration. Because embryonic cells have a high potential for growth and differentiation, their transplantation should provide a suitable environment capable of promoting and supporting growth of the lesioned adult central or peripheral nervous system. Fetal cell implants, particularly neurons and muscle fibers implanted into the nervous system, possess the ability to induce axons to grow with a strong attraction to the grafted fetal tissue. Much of the published work relating to transplantation has focused on attempts to induce regeneration in the CNS. The results from a large number of investigators have shown that, unlike the previous assumptions of past decades, parts of the CNS are indeed capable of limited regeneration. Both functional and morphological data suggest that the injured brain and spinal cord can recover a certain degree of function following trauma. The contribution of the graft to the reconstruction of the host CNS is not well established. For example, the role of collateral sprouting from undamaged fibers of the host into the damaged region containing the graft has not yet been determined. A major determinant of the extent of regeneration is the environment encountered by the regrowing axons. The mechanisms underlying the axonal growth, guidance, and maturation appear to be strongly influenced by trophic factors in the environment which are appropriate and necessary for regeneration and referred to in the art as "growth-promotors". These substances may be specific for a particular target tissue and are likely to have a wide spectrum of growth-promoting potencies.
The reason a moderate or severe injury to a mammalian nerve may not lead to appropriate innervation of the target tissue may be the proportionally greater distance (due to Wallerian degeneration) those axons must travel. Presumably, trophic substances from nearby tissues may have a greater trophic potential than the intended target tissue. As a result, gap length of an injury has been shown in the prior art to be a primary factor in determining the success of functional regeneration. This lack of target specificity has been suggested in the literature as the underlying cause of the frequent formation of neuromas, as well as the inappropriate contact on other tissues. Thus, significant improvements have been observed after neural anastomoses have been made. The current method of choice in neurosurgical repair of damage to peripheral nerves is simple anastomosing of the cut end of the nerve, although this intervention is limited.
Simple anastomosing will not be sufficient in those circumstances where damage and degeneration is so extensive that the distance between the remaining proximal and distal stumps is excessive. An alternative solution employs the use of a structure or "bridge" across the gap length from the cut proximal stump to either the distal portion of the nerve or to the target tissue itself. In animal studies, the various materials which have been used to bridge the gap include peripheral nerve grafts, mesothelial chambers, millipore snd silicone tubes.
Of particular interest are the artificially produced and commercially available "nerve cuffs" or "nerve guide tubes" which are implanted and extend between the stumps. Prior art nerve guide tubes are electrically passive, that is, do not include any electrical current, and have the shape of a hollow cylinder. The nerve guide tubes are generally made of either silicon or bioresorbable substances. The nerve guide tubes can be filled or coated with a growth supporting matrix such as laminin that promotes neural growth over greater distances than the unmodified nerve guide tube alone. The nerve guide tubes have been studied, reported in the literature and are commercially available. During the regenerative process, between two and three weeks after injury, the host body usually causes the interior of the nerve guide tube to fill with a viscous fluid containing proteins and other material in an amorphous matrix. Protein strands appear oriented along the longitudinal axis of the chamber, and may serve as the substrate for cellular migration. Before axons appear, Schwann cells and fibroblasts infiltrate the matrix. In general, blood vessels appear last, although some studies have observed capillary formation prior to axonal growth. It has been suggested that the early invading cells modify the matrix of the nerve guide tube and thereby facilitate the ingrowth of axons. It is important to note that the extracellular matrix will form in the absence of a distal segment. No axonal outgrowth will occur, indicating that the matrix is by itself insufficient to promote axonal growth. Perhaps the distal stump provides a humoral agent diffusible in the matrix which is necessary for growth and/or guidance of axons. This would be similar to the requirement for Schwann cell or muscle cell "conditioned media" in the growth of sensory, sympathetic or motor neurons in vitro. Axon diameter and density are greater when a distal stump is present. Whatever the exact mechanisms responsible for growth, (structural, cellular, and/or humoral), and wherever the site of action (at the axon or its substratum), the nerve guide tubes provide an "artificial" environment suitable for supporting axonal growth over relatively long distances.
As used herein, a "nerve guide means" is an electrically passive physical structure for enhancing nerve regeneration and includes prior art nerve guide tubes and other structures which are not tubular such as a plate-shaped object, solid tubes and other shapes which are effective for enhancing nerve regeneration.
Some cell types have been shown to be affected by static and dynamic electromagnetic fields. Most extensively studied are the effects of electromagnetic fields on bone growth, and prior art reports of in vitro results have demonstrated beneficial effects to some specific types of cells. Recently, published studies in bone-derived cell cultures have shown that electromagnetic fields induce specific biochemical alterations. such effects include cAMP fluctuations, altered states of actin polymerization, enhanced DNA synthesis and changes in calcium uptake. The exact mechanisms responsible for electromagnetic field induced bone growth have not been characterized fully. Due to the complex morphology of the neuronal cell, as well as its ability to grow in vitro, the nervous system is particularly well-suited for studies of the effect of electromagnetic fields on the growth of cells. In the past ten years, many studies have demonstrated that neurite elongation and orientation can be influenced by an electromagnetic field. Specifically, within a static electric field, neurite growth is directed toward the cathode. Changes in the orientation of these neurites can be observed with light microscopy after a period of time from about several minutes to about several hours. All neurons that have been studied to date in vitro respond in some way to an applied electromagnetic field. Many variations in electromagnetic field parameters have been used to observe changes in neurite growth.
The vigorous in vitro response of all neuronal cell types to a wide range of electromagnetic field effects (and thus an apparent lack of specificity for cell type or stimulus) has been used as an argument against the concept that endogenous fields serve a primary role in the guidance of growing neuronal processes in vivo. This is often supported by studies showing neurite growth to a target in the absence of intrinsic action potentials, and axonal synaptogenesis occurring during the blockade of postsynaptic ion channels. Growth and guidance in the nervous system is complex. The local microenvironment with respect to the events required for growth and guidance must necessarily be important for the establishment of proper channels to the target cell. These events are multifactorial, and include a variety of bioelectrochemical processes, such as the timing of membrane interactions between growing axons and glia. It is likely that such multiple interactions are subtle, and may require extremely small local electrical interactions. Action potentials or postsynaptic events may therefore be insufficient alone, or temporarily inappropriate to affect the growth process significantly.
The method of application of the electromagnetic fields used in the in vitro studies is vastly different from what would be possible under local microenvironmental conditions. Typically, in vitro studies apply an electromagnetic field across a large population of cells, often in a culture dish having a volume of enormous size in comparison to the cell, and with a concomitantly applied homogenous current density. Experimental results substantiate that electromagnetic fields may serve a modulatory function in orienting neurite growth within localized regions.
The application of extracellular direct current electric fields in vitro may accelerate as well as orient the growth of neurites in embryonic explants or in dissociated neuronal cultures. The mechanisms by which these biochemical alterations occur are not well understood, nor are they necessarily directly related. In addition, different neuron types appear to respond differently with regard to stimulation amplitude and duration. The biophysical mechanism responsible may be related to the electrophoretic redistribution of cytoplasmic components which may occur if an extracellular potential produces a voltage potential drop in the cytoplasm. The site of most of the cell's electrical resistance is the plasma membrane so that the electromagnetic field would be the strongest and have the greatest voltage potential difference relative to the cytoplasm. Thus, an electromagnetic field may alter the membrane's voltage potential asymmetrically, thereby perturbing growth-controlling transport processes across the membrane. The cytoplasm has far less resistivity than the plasma membrane, and the voltage potential drop is on the order of 10.sup.-4 volts. The majority of work published involving electromagnetic fields on whole cells in vitro use a static electromagnetic field in the range of 0.1 to 15 V/cm, roughly translating into an average of 10 mv/cell diameter. Assuming that 50% of this voltage is exerted across the plasma membrane at each end of the cell, this would result in a hyperpolarization or depolarization of 5 mv, depending on the polarity. Most neuronal resting membrane potentials are approximately -70 to -90 mv and local ion conductances or enzyme activation states at the membrane may be changed enough to alter or modify the normal function.
Furthermore, an electrophoretic accumulation of molecules responsible for neuritic extension and/or adhesion may occur toward the membrane. A charged macromolecule of ordinary electrophoretic mobility (1 micron/sec/V/cm) across a 10 micron distance requires 10.sup.4 to 10.sup.6 seconds (three hours to ten days). It is possible that higher electromagnetic field strengths (approximately 10 V/cm) can cause substantial intracellular migration of growth-related molecules, or receptors for trophic substances. It has been shown in the prior art that the accumulation of surface glycoproteins can occur electrophoretically at the cathode in isolated cultured cells. Membrane glycoproteins are believed to play a crucial role in cell adhesion to the substratum. Cathodal accumulation of these molecules at the membrane may be responsible for some of the orienting effects of the electromagnetic field. These hypotheses are consistent with the majority of the prior art data showing that most changes in directionality or growth rate occur within twenty-four hours of exposure in vitro. Thus, an electromagnetic field in vitro produces a growth promotion effect as well as a guidance effect.
U.S. Pat. No. 4,306,561 discloses methods and apparatuses for the reattachment and repair of severed nerves in a human body. The '561 Patent describes the use of direct current stimulation of the nerve from the proximal nerve end to the distal nerve end to evoke an action potential (i.e. transmittance of electrical activity in the nerve) to test electrical continuity across the two juxtaposed nerve ends. The '561 patent does not, however, suggest the use of electric current as a means for regeneration of nerves. Further, the '561 patent strongly discourages suturing nerve ends together. The '561 patent discloses a device for holding the nerve ends in abutment which requires vacuum lines to engage the nerve ends and is generally in the form of a modified forceps. Thus, the device is not at all suitable for being implanted and the disclosure limits its use to a period of about 5 hours because the patient has an open wound during the use of the device. The requirement disclosed in the '561 patent that the nerve ends abut each other precludes the regeneration of a damaged nerve for which the nerve ends are spaced apart.
From the background given above, it can be appreciated that mammalian nerve regeneration and particularly peripheral nerve regeneration is a complex phenomenon. Furthermore, it can present serious problems to the neurosurgeon who wishes to intervene in some way to increase the chances of good functional recovery following severe damage to nerves, particularly to peripheral nerves.